JP4577873B2 - Methods for improving reagent stability - Google Patents

Description

The present invention relates to a dye stabilization method and composition, and further to a glycated protein measurement method and composition using the same, and more particularly to a dye stabilization method, glycated protein measurement method and quantitative composition useful for clinical examination.

Measurement of glycated protein is very important in the diagnosis and management of diabetes. Glycated hemoglobin (GHb), which reflects the average blood glucose level in the past about 1 to 2 months, and glycation, which reflects the average blood glucose level in the past about 2 weeks Albumin (GA), fructosamine (FRA), which is a general term for glycated proteins exhibiting reducing ability in serum, and the like are routinely measured. In GHb, the α-amino group of the β-chain N-terminal valine of hemoglobin is glycated, and in GA and FRA, albumin and the ε-amino group of the lysine residue of serum protein are glycated.
Enzymatic methods (Patent Documents 1 to 6 and Non-Patent Document 1) can be cited as methods for quantifying glycated proteins that are highly accurate, simple and inexpensive. However, there are few reports on the liquid composition for measuring glycated protein, and Patent Document 7 only describes the stabilization of enzymes that act on proteases, particularly proteases and glycated amino acids.
On the other hand, chromophoric dyes that are commonly used, especially Trinder type dyes, are stable in a dye-only solution, and it has been known that these chromophoric dyes have become unstable in glycated protein measurement reagents. It was not done. Therefore, of course, there was no report about the stabilization of the dye which became unstable in the glycated protein measurement reagent.
Furthermore, Patent Document 8 describes a kit characterized by coexisting with 200 mM or more of electrolyte for the purpose of increasing the fructosyl valine specificity of fructosylamine oxidase when measuring hemoglobin A1c. As a peroxidase to 4 aminoantipyrine system. However, Patent Document 8 has no description on the stability in the liquid state, regardless of the stabilization of the reagent and the dye. Further, there is no detailed description of the combination with protease. A dye stabilization method and composition characterized in that the number of moles of the buffer is 50 or more when the number of moles of the dye is 1 have not been known so far.

JP-A-6-46846 Japanese Unexamined Patent Publication No. 5-192193 Japanese Unexamined Patent Publication No. 2-195900 Japanese Patent Laid-Open No. 2-195899 WO98 / 48043 Publication WO97 / 13872 Publication WO02 / 061119 Publication JP 2001-204494 A Kouzuma et.al.An enzymatic method for the measurement of glycated albumin in biological sample.Clinica Chimica Acta 324 (2002) 61-71

An object of this invention is to provide the method and composition which improve the stability of the pigment | dye used for a clinical test, Furthermore, the glycated protein measuring method and composition using the same.

The present inventor has so far carried out stabilization of enzymes acting on proteases and glycated amino acids, in particular for the purpose of developing a liquid and stable glycated protein measuring reagent. However, the liquid glycated protein measurement reagent was found to have insufficient storage stability during refrigerated storage only by stabilizing the enzyme.
Thus, as a result of examining components other than the enzyme that contributes to the stability, it was found that there is a cause that the reagent deteriorates due to the denaturation of the coloring dye, particularly 4-aminoantipyrine. Since 4-aminoantipyrine is generally liquid and stable, it was considered to be an unexpected phenomenon that occurs in a special environment such as when it is contained in a glycated protein measurement reagent.
Therefore, as a result of diligent study of the stabilization of the dye, the present inventors have found that the stabilizing effect is small with a normal stabilizer, and surprisingly, the dye stability is effectively increased by increasing the concentration of the buffer. The present invention has been found. It was also found that the reagent can be more efficiently stabilized by further increasing the concentration of 4-aminoantipyrine to 1.0 mM or more.

That is, the present invention
1) A composition containing a dye and a buffer, wherein the number of moles of the buffer is 50 or more when the number of moles of the dye is 1.
2) The composition according to 1), further comprising an enzyme.
3) The composition according to 1) or 2), which is liquid.
4) The composition according to 3), wherein the concentration of the buffer is 100 mM or more.
5) The composition according to 3) or 4), wherein the concentration of the dye is 1.0 mM or more.
6) The composition according to any one of 1) to 5), wherein the buffer is 3,3 dimethyl glutaric acid.
7) The composition according to any one of 1) to 6), wherein the dye is 4-aminoantipyrine.
8) A composition comprising an enzyme, a dye and a buffer, wherein the dye is stable for more than 6 months when refrigerated.
9) A method for improving the stability of a dye with a buffer, wherein the number of moles of the buffer is 50 or more when the number of moles of the dye is 1.
10) A method for improving the stability of a dye with a buffer in the presence of an enzyme, wherein the number of moles of the buffer is 50 or more when the number of moles of the dye is 1. How to improve.
11) The method according to 9) or 10), wherein the concentration of the buffer is 100 mM or more.
12) The method according to any one of 9) to 11), wherein the concentration of the dye is 1.0 mM or more.
13) The method according to any one of 9) to 12), wherein the dye is 4-aminoantipyrine.
14) The method according to any one of 9) to 13), wherein the buffer is 3,3 dimethyl glutaric acid.
15) Method for measuring glycated protein using any one of compositions 1) to 8)

The method and composition for improving the stability of the present invention has an effect of stabilizing a dye, and particularly has an effect of stabilizing a reagent used for measurement of a glycated protein.

The present invention will be specifically described below.
As the dye that can be used in the present invention, any dye may be used as long as it can be used for glycated protein measurement using an enzyme. For example, a reducing dye such as a reduced coenzyme or a tetrazolium compound, Oxidizing dyes such as Trinder dyes and leuco dyes can be used. Among them, Trinder dyes and leuco dyes are preferred, more preferred are tender dyes, and among the tender dyes, 4-aminoantipyrine is preferred. Most preferred.
The Trinder type dye consists of a hydrogen donor and a coupler. In the present application, the term “dye” includes both a hydrogen donor and a coupler in the concept of “dye”.
Any coupler can be used as long as it condenses with a hydrogen donor and develops color. For example, 4-aminoantipyrine or 3-methyl-2-benzothiazolinone hydrazone (MBTH) can be used.

As the hydrogen donor of the Trinder type reagent, phenol derivatives, aniline derivatives, toluidine derivatives, etc. can be used. Specific examples include sodium N- (3-sulfopropyl) aniline 1 water (HALPS), N-ethyl-N. -(3-sulfopropyl) -3 methylaniline sodium 1 water (TOPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium 1 water (MAOS), N- (3-sulfopropyl) -3,5-dimethoxyaniline sodium 1 water (HDAPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium (HDAOS), N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxyaniline sodium 1 water (DAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium (DAOS), N- Ethyl-N- (3-sulfopropyl) aniline sodium (ALPS), N-ethyl-N- (3-sulfopropyl) -3- Toxianiline sodium 1 water (ADPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline sodium 2 water (ADOS), N-ethyl-N- (2-hydroxy-3- Sulfopropyl) -m-toluidine (TOOS), N, N bis (4-sulfobutyl) -3-methylaniline disodium (TODB; manufactured by Dojindo Laboratories) and the like.

Specific examples of the leuco reagent include N- (carboxymethylaminocarbonyl) -4,4-bis (dimethylamino) biphenylamine (DA64), 10- (carboxymethylaminocarbonyl) 3,7-bis (dimethylamino). ) Phenothiazine (DA67), 2,2'-aminobis (3-ethylbenzothiazolinone-6-sulfonic acid (ABTS), bis- (4-diethylaminophenyl) -2-sulfophenylmethane (BSMP), bis [ 3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA); manufactured by Wako Pure Chemical Industries, Ltd., 3,3′-diaminobenzidine · 4HCI (DAB), 3- (4-hydroxyphenyl) propion Acid (HPPA), N, N'-bis (2-hydroxy-3-sulfophenyl) tolidine.2Na.4H ₂ O (SAT-3), 3,3 ', 5,5'-tetramethylbenzidine (TMBZ ), N- (3-sulfopropyl) -3,3 ', 5,5'-tetramethylbenzidine Na (T MBZ-PS), N, N ′, N ′, N ″, N ″ -hexa (3-sulfopropyl) -4,4 ′, 4 ″ -triaminotriphenylmethane · 6Na (TPM-PS) Doujin Chemical Laboratory Co., Ltd.).
Tetrazolium compounds include nitrotetrazolium blue, tetrazolium blue, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4disulfophenyl) -2H-tetrazolium: WST-1, 2- (4-Iodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4disulfophenyl) -2H-tetrazolium: WST-3, 2- (2-methoxy-4-nitrophenyl)- 3- (4-Nitrophenyl) -5- (2,4disulfophenyl) -2H-tetrazolium: Various tetrazolium salts represented by WSR-8 (manufactured by Doujin Chemical Laboratory Co., Ltd.) can be used.

Any concentration of these dyes may be used as long as the glycated protein can be measured. For example, the lower limit is 1.0 mM or more, preferably 1.5 mM or more, and the upper limit is 500 mM or less. Is 300 mM or less.
As the buffering agent that can be used in the present invention, any buffering agent may be used as long as the pigment is stable, or a buffering agent that stabilizes the pigment in the presence of the enzyme and the pigment.
Specific examples of buffering agents include, for example, Good's buffering agents such as 3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (EPPS), 2- [4- (2-hydroxyethyl) ) -1-Piperazinyl] ethanesulfonic acid (HEPES), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid (HEPPSO), N- (2-acetamido) -2- Aminoethanesulfonic acid (ACES), N- (2-acetamido) iminodiacetic acid (ADA), N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), N, N-bis (2 -Hydroxyethyl) glycine (Bicine), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), N-cyclohexyl-2-hydroxy- 3-aminopropanesulfonic acid (CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3- [N, N-bis (2-hydrido Roxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), 2-hydroxy-3-morpholinopropanesulfone Acid (MOPSO), Piperazine-1,4-bis (2-ethanesulfonic acid (PIPES), Piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO), N-Tris (hydroxymethyl) ) Methyl-3-aminopropanesulfonic acid (TAPS), 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPSO), N-tris (hydroxymethyl) methyl-2-aminopropanesulfone Acid (TES), N- [Tris (hydroxymethyl) methyl] glycine (Tricine) and Trishydroxymethylaminomethane (Tris), N- (2-acetamido) -2-aminoethanesulfonic acid (ACES), N- ( 2-acetamido) iminodiacetic acid (ADA) etc. Impactant, or boric acid, ammonia, glycine, carbonic acid, acetic acid, phosphoric acid, diethanolamine, p-phenolsulfonic acid, 2-amino-2-methylpropane-1,3-diol, cacodylic acid, citric acid, maleic acid, Examples include veronal and 3,3 dimethyl glutaric acid.

Examples of preferable buffering agents include buffers having a buffering action near pH neutrality, such as EPPS, HEPES, HEPPSO, ACES, ADA, BES, Bicine, Bis-Tris, CHES, DIPSO, MES, MOPS, MOPSO, PIPES. , POPSO, TAPS, TAPSO, TES, Tricine, Tris, ACES, ADA, boric acid, ammonia, glycine, phosphoric acid, diethanolamine, p-phenolsulfonic acid, 2-amino-2-methylpropane-1,3-diol, Examples thereof include cacodylic acid, citric acid, maleic acid, veronal and 3,3 dimethyl glutaric acid, and most preferably 3,3 dimethyl glutaric acid.
Any concentration of these buffers may be used as long as the dye is stable. For example, the lower limit is 100 mM or more, preferably 150 mM or more, and the upper limit is 5.0 M or less, preferably 1 It may be used at a concentration of 0 M or less.

As a composition that can be used in the present invention, a known composition, preferably a composition for measuring glycated protein, and a combination of the dye and buffer of the present invention may be used. In particular, a combination of an enzyme, a dye and a buffer Is preferred.
As the enzyme that can be used in the present invention, any enzyme may be used, but it is preferably an enzyme necessary for the measurement of glycated protein, such as protease, ascorbate oxidase, enzyme acting on glycated amino acid, and peroxidase. And preferably a protease and an enzyme that acts on glycated amino acids, and the most preferred enzyme is a protease.
As the protease that can be used in the present invention, any protease can be used as long as it effectively acts on a glycated protein contained in a test solution and effectively produces a glycated amino acid and / or a glycated peptide derived from the protein. Good, for example, animal, plant-derived proteases, genus Bacillus, Aspergillus, Rhizopus, Penicillium, Streptomyces, Staphylococcus, Clostridium And proteases derived from microorganisms such as Lysobacter, Glifila, Yeast, Tritirachium, Thermus, Pseudomonus, Achromobacter, and the like. Examples of proteases derived from microorganisms belonging to the genus Bacillus include subtilisin and metalloprotease.

  In addition, when the glycated protein to be measured is glycoalbumin, microorganism-derived proteases of the genus Bacillus and Streptomyces are more preferable because they have a large effect on human albumin. Also, when the glycated protein to be measured is glycated hemoglobin, proteases derived from the genera Bacillus, Aspergillus, Streptomyces, and Trityratium are more preferable because they have a large effect on human hemoglobin.

  The protease activity measurement method that can be used in the present invention was measured using the casein-forin method.

As an enzyme that acts on a glycated amino acid that can be used in the present invention, by the action of the protease, it effectively acts on a glycated amino acid or a glycated peptide produced from a glycated protein contained in a test solution. Any enzyme can be used as long as it can be measured, but preferably, an enzyme that acts on a glycated amino acid that works well on an amino acid having an α-amino group, and a glycation that works on an amino acid in which an ε-amino group works. Examples include enzymes that act on amino acids.
Examples of enzymes that act on glycated amino acids that have a strong effect on glycated amino acids in the ε-amino group include the genus Gibberella, the genus Aspergillus, the genus Candida, the genus Penicillium, and the Fusarium. Enzymes from the genera, Acremonium or Debaryomyces.
An example of an enzyme that acts on a glycated amino acid that frequently acts on an amino acid whose α-amino group has been glycated is an enzyme derived from Corynebacterium.
Furthermore, examples of enzymes that have sufficient activity even in the presence of proteases and can be produced at low cost include genetically modified ketoamine oxidase (KAOD; manufactured by Asahi Kasei Pharma; described in Non-Patent Document 1) and In addition, a mutant KAOD (KAOD-V; manufactured by Asahi Kasei Pharma Co., Ltd.) containing a FOD gene described in Patent Document 2 is produced that has significantly reduced glycated valine reactivity. KAOD). The activity of the enzyme acting on the glycated amino acid was measured by the method described in Non-Patent Document 1.

Any concentration of these enzymes may be used as long as the glycated protein can be measured. For example, in the case of protease, the lower limit is usually 10 U / ml or more, preferably 100 U / ml or more, most preferably. Is 500 U / ml, the upper limit is 1.0 × 10 ⁶ U / ml or less, preferably 5.0 × 10 ⁵ U / ml or less, and most preferably 3.0 × 10 ⁵ U / ml or less. It is. For example, in the case of ketoamine oxidase, ascorbate oxidase and peroxidase, the lower limit is usually 0.5 U / ml or more, preferably 1 U / ml or more, and the upper limit is 1000 U / ml or less, preferably 500 U / ml or less. is there.

As the combination of the dye and the buffer that can be used in the present invention, any combination may be used, and the aforementioned dye and buffer may be used. The concentration ratio of the dye to be combined and the buffer may be any concentration ratio as long as the dye is stable for refrigerated liquid storage for 6 months or more, preferably 9 months or more. For example, the number of moles of the dye is 1. In this case, the number of moles of the buffering agent is preferably 50 or more, and more preferably, the number of moles of the buffering agent when the number of moles of the dye is 1 is 100 or more.
As the method and composition for measuring glycated protein of the present invention, any method and composition may be used as long as it is a method and composition for measuring glycated protein using the stability improving method of the present invention. Furthermore, as the composition for measuring glycated protein of the present invention, a composition comprising a proteolytic reagent containing a protease and a detection reagent containing an enzyme that acts on a glycated amino acid can be used. Is contained so that the number of moles of the buffer is 50 or more when the number of moles of the dye is 1, or the protease, the enzyme acting on the glycated amino acid, the dye and the buffer have the mole number of the dye of 1 In this case, the number of moles of the buffering agent may be included at the same time so as to be 50 or more. These reagents can be provided as a liquid product, a frozen product of the liquid product, or a freeze-dried product, but a preferred dosage form is a liquid product. Examples of the liquid product include an aqueous solution containing a dye and a buffer, and an aqueous solution containing a dye, a buffer and an enzyme.

Among the specific glycated proteolytic reagent compositions that can be used in the present invention, as the proteolytic reagent, the pH, buffer and protease concentration are determined so that the proteolytic reaction proceeds efficiently, and then ASOx, protease stable What is necessary is just to prepare and add a chemical | medical agent etc. suitably so that it may become effective concentration.
For example, when protease type XXIV (manufactured by Sigma) is used, since the proteolytic activity is strong around pH 7 to 10, the pH of the reaction can be selected from 7 to 10. As the dye, 4-aminoantipyrine can be used, and the use concentration is, for example, a lower limit concentration of 0.2 mM or more, preferably 0.3 mM or more, and an upper limit concentration of 1.0 M or less, preferably 0.500 mM or less. do it. In addition, 3,3 dimethyl glutaric acid can be used as a buffer for stabilizing the dye, and the number of moles of 3,3 dimethyl glutaric acid is 50 or more when the number of moles of 4 aminoantipyrine is 1. Use to be. At this time, the concentration of 3,3 dimethyl glutaric acid is preferably 100 mM or more, for example, 200 mM.
As a protease stabilizer, a known stabilizer may be used.For example, when DMSO is added as a stabilizer, the lower limit concentration is 5% or more, preferably 10% or more, and the upper limit concentration is It may be added at a concentration of 50% or less, preferably 40% or less.
Regarding the glycated amino acid quantitative reagent composition that can be used in the present invention, the pH is selected so that the reaction proceeds efficiently in consideration of the optimum pH of the enzyme that acts on the glycated amino acid to be used, and then the enzyme that acts on the glycated amino acid What is necessary is just to add the stabilizer of the enzyme which determines quantity and finally acts on a glycated amino acid.

For example, when using the KAOD or KAOD-V (manufactured by Asahi Kasei Pharma), the region showing an activity of 50% or more of the maximum activity is as wide as pH 6.5 to 10, and the reaction pH can be selected from 6.5 to 10.
As an enzyme stabilizer that acts on glycated amino acids, for example, sorbitol can be added, the lower limit concentration is 0.01% or more, preferably 0.1% or more, and the upper limit concentration is 30% or less, preferably 20% or less. It may be added at a concentration of.
Furthermore, for example, surfactants, salts, preservatives and the like may be appropriately selected and added to the enzyme reaction composition for quantifying the glycated protein based on the present invention.
As the surfactant, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyvinyl alcohol, etc., 0.01 to 10%, preferably 0.05 to 5%, various metal salts such as lithium chloride, sodium chloride, chloride 1 mM to 5 M of potassium, manganese chloride, cobalt chloride, zinc chloride, calcium chloride, etc., preferably 10 mM to 1 M, various preservatives, for example, 0.01 to 10%, preferably 0.05 to 1% of sodium azide Just do it.

  In the present invention, the stability of the dye is determined, for example, as follows. If the composition containing the dye and the buffer is not a liquid product, for example, if it is a frozen product or a freeze-dried product, it is made into a liquid product by thawing or adding water. In the determination of stability, the liquid product is adjusted so that the pigment concentration contained in the liquid product is a pigment concentration applicable to glycated protein concentration measurement. This adjusted liquid product (in the case of being marketed in the form of a liquid product so that it can be used as it is for the measurement of glycated protein concentration without adjustment, it means that the container filled with the liquid product is opened) Refrigerated (2-10 ° C.) storage, preferably 4-6 ° C., more preferably 5 ° C., for 1 year (alternatively, 37 ° C., 1 week storage conditions may be substituted). Using this liquid product after storage and the liquid product immediately after adjustment, glycated albumin or glycated hemoglobin is measured, the sensitivity of the liquid product after storage and immediately after adjustment is measured, and the decrease in sensitivity is calculated. When this decrease in sensitivity is suppressed, it is said that stability is improved in the present invention. In the present invention, the term “stable dye” means that the retention of sensitivity is 80% or more, preferably 90% or more when the same liquid sample after storage and immediately after preparation is measured.

As a method for measuring a glycated protein that can be used in the present invention, 0.001 to 0.5 ml of a test solution is added to the composition for quantifying glycated protein of the present invention and reacted at a temperature of 37 ° C. to perform a rate assay. Is changed coenzyme in a few minutes to several tens of minutes after a certain time after the start of the reaction, for example, 1 minute after 3 minutes and 4 minutes, or 5 minutes after 3 minutes and 8 minutes, The amount of dissolved oxygen, hydrogen peroxide or other products may be measured directly or indirectly by the above method. In the case of an endpoint assay, the changed coenzyme, dissolved oxygen, The amount of hydrogen peroxide or other products may be measured in the same way. In this case, the amount of glycated protein in the test solution can be determined by comparing with changes in absorbance or the like when measured using a glycated protein at a known concentration.

In addition, as a method for measuring a glycated protein of the present invention, the glycated protein ratio can be obtained by separately quantifying the protein. For example, if the protein is glycated albumin, albumin may be measured separately, and if the protein is glycated hemoglobin or hemoglobin A1c, hemoglobin may be measured separately. Measurement of albumin and hemoglobin may be performed using a known method.
As the test solution to be measured in the present invention, any test solution containing at least glycated protein may be used. However, preferred test solutions include blood components such as serum, plasma, blood cells, and whole blood. Examples include blood. Separated erythrocytes can also be used as a preferred test solution because the globulin component may be mixed depending on the separation conditions and affect the measured value.
Examples of the glycated protein to be measured in the glycated protein quantitative composition and method of the present invention include glycated albumin or glycated hemoglobin (hemoglobin A1c), but the glycated protein to be measured is not limited to these. Any glycated protein may be measured.

The present invention will be described based on examples.
[Example 1]
Stabilized composition for measuring glycated albumin
R-1 Proteolytic reagent
150 mM 3,3 dimethyl glutaric acid (manufactured by Wako Pure Chemical Industries, Ltd.)
8000U / ml protease type XXIV (manufactured by Sigma)
2.0 mM 4-aminoantipyrine (Wako Pure Chemical Industries, Ltd.)
20% dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.)
R-2 Glycated amino acid detection reagent
150 mM Tricine buffer (Wako Pure Chemical Industries, Ltd.) pH 7.5
3.0 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS, manufactured by Wako Pure Chemical Industries, Ltd.)
15U / ml KAOD (Asahi Kasei Pharma)
20 U / ml peroxidase (Sigma)
5% Sorbitol (Wako Pure Chemical Industries)

[Example 2]
Stabilized composition for measuring glycated hemoglobin
R-1 Proteolytic reagent
150 mM 3,3 dimethyl glutaric acid (manufactured by Wako Pure Chemical Industries, Ltd.)
8000U / ml protease type XIV (manufactured by Sigma)
2.0 mM 4-aminoantipyrine (Wako Pure Chemical Industries, Ltd.)
2.0 mM 2- (4-iodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4disulfophenyl) -2H-tetrazolium: WST-3 (manufactured by Dojindo Laboratories)
20% dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.)
R-2 Glycated amino acid detection reagent
150 mM Tricine buffer (Wako Pure Chemical Industries, Ltd.) pH 7.5
3.0 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS, manufactured by Wako Pure Chemical Industries, Ltd.)
15U / ml KAOD (Asahi Kasei Pharma)
20 U / ml peroxidase (Sigma)
5% Sorbitol (Wako Pure Chemical Industries)

[Example 3]
Effect of buffer concentration on reagent stability
The composition for measuring glycated protein described in Examples 1 and 2 was stored in a refrigerator (about 5 ° C.) for 1 year. The sensitivity of the control was measured immediately after preparation and one year after refrigerated storage.
<Reaction procedure of glycated albumin measuring reagent>
8 μl of control serum (manufactured by BML Co., Ltd.) was added to 240 μl of R-1 incubated at 37 ° C., the reaction was started at 37 ° C., and R-2; 80 μl was added exactly 5 minutes later. Absorbance at 555 nm was measured before the addition of R-2 and 5 minutes after the addition. Further, a measurement using distilled water instead of the substrate solution was used as a blank, and ΔA ₀ was calculated by subtracting the absorbance change of the blank sample from the absorbance change obtained from the control substrate solution.
<Reaction procedure of glycated hemoglobin measuring reagent>
Add 30 μl of HbA1c real sample standard material (primary calibrator JDS HbA1c Lot2 Welfare and Medical Technology Promotion Association) to R-1; 240 μl incubated at 37 degrees, start reaction at 37 ° C, and accurately 5 After 60 minutes, R-2; 60 μl was added. Absorbance at 555 nm was measured before the addition of R-2 and 5 minutes after the addition. Further, a measurement using distilled water instead of the substrate solution was used as a blank, and ΔA ₀ was calculated by subtracting the absorbance change of the blank sample from the absorbance change obtained from the control substrate solution.
As a result of measuring the sensitivity change, assuming that the initial sensitivity is 100%, the remaining sensitivity of the glycoalbumin reagent and the glycated hemoglobin measurement reagent was 85% and 77% after one year of refrigerated storage, respectively. This shows that the composition for measuring glycated protein deteriorated after refrigerated storage for 1 year.

Next, for the purpose of examining which of R-1 and R-2 is the cause of the sensitivity decrease, R-1 and R-2 after storage and newly created R-1 and R-2 are used. Combination of R-1, new R-2, new R-1, combination of R-2 after storage, combination of R-1, R-2 after storage, combination of new R-1, R-2 Control was measured. In the case of a glycated albumin reagent, the measurement results are 85% for the R-1 and R-2 after storage, and the new R-1 stored if the sensitivity when using a combination of new R-1 and R-2 is 100% The relative sensitivity was 98% for the later combination of R-2 and 87% for the combination of R-1 after storage and new R-2. From this, it was found that the component of R-1 was the cause of deterioration.
Furthermore, when the addition experiment of each component of R-1 was conducted for the purpose of ascertaining the cause of deterioration in R-1, the sensitivity recovered only in the case of addition of 4-aminoantipyrine as a dye. This revealed that the R-1 dye was deteriorated.

[Example 4]
Effect of Buffer Concentration on Reagent Stability Glycoalbumin Measurement Reagent R1 Buffer 3,3 Dimethylglutaric acid concentration is changed to 25mM, 50mM, 75mM, 100mM, 200mM, 300mM at 37 ° C for 1 week The control sensitivity was measured after standing. In addition, it is known that the storage test of this reagent at 37 ° C for 1 week corresponds to 1 year of refrigerated storage. The results are shown in FIG.
As can be seen from FIG. 1, it was clear that the reagent was dramatically stabilized by using a buffer of 100 mM or more. This was considered to be the stabilization of the dye in the reagent, in this case 4-aminoantipyrine. In this case, since 2.0 mM 4-aminoantipyrine is used as the dye and the buffer concentration is preferably 100 mM or more, the number of moles of the buffer is preferably 50 or more when the number of moles of the dye is 1. Is obvious.
In addition, when the number of moles of the dye is 1, the sensitivity of the composition having the number of moles of the buffer of 50 or more after storage at 37 ° C. for 1 week is 90% or more, and the GA concentration after calibration is Since there was no change before and after the test, the composition using this stabilization method was considered to be stable in a liquid state for at least 6 months of refrigeration. Further, when the same test as in this study was used for the glycated hemoglobin composition, the reagent having a composition in which the number of moles of the buffer was 50 or more when the number of moles of the dye was set to 1 was dramatically stabilized. .

[Example 5]
Effect on the stability of 4-aminoantipyrine concentration Change the 4-aminoantipyrine concentration of the glycated albumin measurement reagent to 0.5 mM, 1.0 mM, 2.0 mM, 5.0 mM and leave the reagent at 37 ° C for 1 week to measure the sensitivity of the control did. The results showed that the remaining sensitivities were 80%, 93%, 94%, and 95%, respectively, and the stability was high at a 4-aminoantipyrine concentration of 1.0 mM or higher. This is because the phenomenon of reagent deterioration is caused by the deterioration of the dye during storage, and it was thought that if the amount of 4-aminoantipyrine was increased from the beginning, deterioration in reagent performance would decrease. However, increasing the amount of 4-aminoantipyrine alone did not completely stabilize the reagent. As can be seen from this result, when stabilizing the dye, when the mole number of the dye is 1, the buffering agent mole number is 50 or more, and the 4-aminoantipyrine concentration is 1.0 mM or more. Good things were obvious.
Moreover, when the same test as this examination was conducted with the glycated hemoglobin composition, the stability was improved at a 4-aminoantipyrine concentration of 1.0 mM or more.

The stabilization method and composition of the present invention can be suitably used in the field of clinical examination.

It is the figure which showed the buffer agent density | concentration based on Example 4 of this invention, and the relationship of the stability of a pigment | dye.

Claims (10)

A 4-aminoantipyrine stabilizing composition containing 4-aminoantipyrine and 3,3-dimethylglutaric acid, wherein the moles of 3,3-dimethylglutaric acid when the number of moles of 4-aminoantipyrine is 1 A 4-aminoantipyrine stabilizing composition, wherein the number is 50 or more. The 4-aminoantipyrine stabilizing composition according to claim 1, further comprising an enzyme. The 4-aminoantipyrine stabilizing composition according to claim 1 or 2, wherein the composition is liquid. The 4-aminoantipyrine stabilizing composition according to any one of claims 1 to 3, wherein the concentration of 3,3-dimethylglutaric acid is 100 mM or more. The 4-aminoantipyrine stabilizing composition according to any one of claims 1 to 4 , wherein the concentration of 4-aminoantipyrine is 1.0 mM or more. A method for improving the refrigerated storage stability of 4-aminoantipyrine in a liquid composition by containing 3,3-dimethylglutaric acid, wherein the number of moles of 4-aminoantipyrine is 1, A 4-aminoantipyrine stability improving method, wherein the number of moles of 3-dimethylglutaric acid is 50 or more. The method for improving 4-aminoantipyrine stability according to claim 6 , which is a method for improving the refrigerated storage stability of 4-aminoantipyrine in the presence of an enzyme by containing 3,3-dimethylglutaric acid. The method for improving stability of 4-aminoantipyrine according to claim 6 or 7 , wherein the concentration of 3,3-dimethylglutaric acid is 100 mM or more. The method for improving stability of 4-aminoantipyrine according to any one of claims 6 to 8 , wherein the concentration of 4-aminoantipyrine is 1.0 mM or more. The method to measure glycated protein using the composition in any one of Claims 1-5 .

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